DESCRIPTION: Metastasis is the major cause of death in breast cancer patients. During metastasis circulating tumor cells (CTCs) disseminated from the primary tumor circulate through the bloodstream and colonize distal tissues. CTC cell count in blood is used as an independent prognostic marker for breast cancer patients, but the heterogeneity and low abundance of CTC have prevented us from unraveling their full diagnostic potential. Recent studies suggest that subpopulations of CTCs can create metastatic lesions. These invasive CTCs are called metastasis-initiating cells (MICs). One promising application of CTCs would be the monitoring of metastasis by detecting MICs through liquid biopsies - a simple, non-invasive cytopathological analysis of CTCs from real-time blood draw. However, current technologies fail to identify MICs owing to biased surface marker selection. The inability to study multiple protein markers in each CTC limits our understanding of MICs. One characteristic of MICs is an invasive phenotype that allows them to extravasate through the endothelial layer of blood vessels. However, current tools used for correlating aggressive cancer cell phenotype (transwell endothelial invasion assays) with proteotype (slab-gel western blot) are not suitable for CTC analysis, given the cell-to-cell heterogeneity and low abundance of CTCs. A novel assay is needed to first identify MICs from CTCs, and then characterize molecular markers unique to MICs. In this 2-year Exploratory/Developmental Research (R21) project, we propose to create a new platform for direct measurement of both (1) transendothelial migration (TEM; phenotype) and (2) metastasis-related signaling (proteotype) in each CTC across populations of these sparingly limited cells. We will advance our recently developed first-in-kind single-cell western blot (scWestern) to include a micro-scale transwell assay (Transwell), allowing direct correlation of biophysical & biochemical responses in single CTCs. We will first optimize the Transwell-scWestern by using breast cancer cell lines expressing a large spectrum of molecular markers. We will then apply our system to CTCs collected from 25 metastatic breast cancer patients, in order to phenotypically select MICs and subsequently profile their metastasis-related signaling state. Direct correlation of MIC phenotype and proteotype will help establish the molecular subtype of MIC. Our study will introduce a novel single-cell CTC biophysical & biochemical assay (Transwell-scWestern) that allows phenotypic selection and subsequent protein analysis. Our long term goal is to provide rapid CTC screening from blood draws based on metastasis-related signaling markers. During this 2 year R21 project, our team will combine efforts from experts in CTC identification and profiling (Jeffrey) and single-cell proteomics (Herr) to advance the vision of liquid biopsies for more accurate diagnosis and precise treatment of breast cancer. Clinical relevance statement: Our long-term goal is identification and molecular classification of metastasis-initiating circulating tumor cells in blood circulation. Sophisticated blood-based cancer assays specifically optimized for early detection of metastasis are needed to manage cancer as a chronic condition, as metastasis is a major cause of cancer mortality.